The present invention relates to an expression and secretion vector which comprises a specific segment of the Bacillus cereus (herein "B. cereus") gene which codes for penicillinase on a plasmid and to the construction of vectors and the use of the vectors in the expression and secretion of one or more exogenous polypeptides in microorganisms for example, B. subtilis.
The term gene as used herein is a chromosomal segment that codes for a single polypeptide chain or RNA molecule. Protein as used herein refers to a macromolecule composed of one or more polypeptide chains, each possessing a characteristic amino acid sequence and molecular weight.
Bacteria are commonly divided into two major categories: gram-positive and gram-negative. The basis for this distinction is the structure of the cell envelope. In gramnegative bacteria, for example, E. coli, the cell envelope consists of three major layers: an inner cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane. Gram-positive organisms, for example, B. subtilis, have a cytoplasmic membrane and a thick cell wall. These different structures affect how the bacteria secretes protein. Because of the three layers in the cell envelope, very few proteins are secreted outside the cell envelope in gram-negative organisms.
The mechanism of transfer of proteins across cell membranes was first described in a eukaryotic system by Blobel, G. and Dobberstein, D., (1975) J. Cell Biol. 67, 835-851. They showed that a protein which is secreted from a cell is synthesized initially as a larger protein precursor with 15-30 additional amino acids at the NH.sub.2 -terminus as compared to the secreted protein. These additional terminal amino acids on the protein are called the "signal peptide" and help direct the transfer of the protein across the cell membrane. The DNA sequence that codes for this peptide is called the "signal sequence". The signal peptide is cleaved from the protein at a specific point during the protein secretion process. Most secreted eukaryotic proteins that have been studied to date have a signal peptide.
In E. coli, a prokaryotic cell, some proteins have also been found to have signal peptides at the NH.sub.2 -terminus. Examples are the maltose-binding protein, the arabinase binding protein, alkaline phosphatase, and .beta.-lactamase; Josefsson, L. and Randall, L. L., (1981) Cell 25, 151-157; Emr, S. D., Hall, M. N. and Silhavy, T. J. (1980) J. Cell. Biol. 86, 701-711. After the protein is processed, in E. coli, these proteins are secreted into the periplasmic space which is between the inner and outer membranes of the cell.
Bacilli and other gram-positive organisms are, however, known to secrete proteins directly into the extracellular medium. The genes for some of the major secretion proteins in Bacilli have already been cloned and DNA sequencing has identified the amino acids in the signal peptide. These include amylase from B. amyloliquefaciens; Palva, I. et al. (1981), B. subtilis; Yang, M., Galizzi, A. & Henner, D., (1983) Nucleic Acids Res. 11, 237-249 Gene 15, 43-51 and penicillinase from B. licheniformis; Neugebauer, K., Sprengel, R. & Schaller, H., (1981) Nucleic Acids Res. 9, 2577-2588. These proteins also have a typical signal peptide that, in the gram-positive Bacilli organism, direct the secreted protein outside the cell.
Cloning of a gene can be defined as a process that involves moving a gene from one cell into a new host's cell, sorting those transformed host cells, and selecting the cells having the particular moved gene from the cell mixture. The gene of interest is moved into a host by means of a vector (carrier) which, for example, can be a large DNA molecule, such as a plasmid. A vector is defined as a DNA molecule known to replicate autonomously in a host cell, to which a foreign segment of DNA may be introduced in order to bring about its replication. A plasmid is defined as an extra-chromosomal, independently replicating small circular DNA molecule. The host and the vector used to clone a gene is referred to as a host vector (Hv) system.
There have been many reports of successful cloning of eukaryotic genes in E. coli, for example, human serum albumin by Lawn, R. M. et al., (1981) Nucleic Acids Res. 9, 6103-6114; calf rennin; Harris, T. J. R. et al., (1982) Nucleic Acids. Res, 10, 2177-2187 and plasminogen activator; Pennica, D. et al. (1983) Nature 301, 214-221. These proteins all have signal sequences and are normally secreted by the host eukaryotic cell. Because the signal peptide from a foreign gene will also be recognized by the E. coli cell, as shown by Talmage, K., Kaufman, J. & Gilbert, W. (1980) Proc. Nat. Acad. Sci. USA 77, 3988-3992 these expressed foreign proteins are found in the periplasm of the E. coli organism if their signal sequence is not removed. This usually makes purification of the secreted proteins more difficult. Therefore, a cloning strategy in E. coli has been developed where the DNA that codes for the signal peptide is removed completely and the gene coding for the mature protein is connected at the 3' end of a bacterial promoter. This has been done with interferon, plasminogen activator and insulin. This results in the accumulation of the foreign protein in the cytoplasm of E. coli, where it can be purified easier than if it accumulated in the periplasm.
The most widely studied gram-positive organism is B. subtilis. It has many advantages over E. coli for the development of a cloning system. Since it is not associated with any human diseases it is a nonpathogen. Unlike E. coli, B. subtilis does not produce endotoxin. This greatly simplifies the purification problems which are inherent in the use of E. coli to produce products for medical or veterinary use. B. subtilis is also a fermentation organism which is grown routinely on an industrial scale. These factors coupled with its potential to secrete useful foreign proteins into the extracellular medium make the organism well suited for a cloning-expression system.
Many B. subtilis cloning vectors have already been established and are described by Gryczan, T. J. (1982) in the Molecular Biology of the Bacilli, Dubnau, D., ed., pp. 307-329, Academic Press, N.Y. which is incorporated herein by reference. These cloning vectors which make it possible to carry new genes to B. subtilis cells, are plasmids which have many unique restriction sites, and encode selectable antibiotic resistance markers which are useful for isolation purposes. The use of these B. subtilis vectors and the optimization of plasmid cloning in B. subtilis as described by Gryczan, T. J., Contente, S. & Dubnau, D. (1980) Mol. Gen. Genet. 177, 454-467 has permitted the cloning of heterologous or foreign genes in B. subtilis. The first report of the cloning of such a heterologous gene in B. subtilis encoded the core antigen (HBV) of hepatitis B and the major antigen (VPI) of foot and mouth disease virus; Hardy, K., Stahl, S. and Kupper, H. (1981), Nature 293, 481-483. Both of these heterologous genes were cloned into the erythromycin gene of pBD9, which is a Bacillus subtilis plasmid chimera with multiple antibiotic resistance; Ehrlich, S. P. et al. (1978) In "Genetic with Multiple Antibiotic Resistance: H. W. Boyer and S. Nicosia, eds., pp. 25-32 and Gryczan, T. J. and Dubnau, D. (1978) Proc. Nat. Acad. Sci. USA 75, 1428-1432. The result of this work was a B. subtilis strain that produced fusion proteins (part erythromycin resistance protein, part viral antigen) in the B. subtilis cell. The erythromycin resistance protein is not a secretory protein.
The first report of the cloning of eukaryotic genes in B. subtilis came from Chang, S. et al., (1982) In Molecular Cloning and Gene Regulation in Bacilli, Ganesan, A. T. et al., ed., pp. 159-169, Acadamic Press, New York. They had previously cloned the penicillinase gene from Bacillus licheniformis (herein "B. licheniformis") in B. subtilis; Gray, O. and Chang, S. (1981) Bacteriol. 145, 422-428. The gene for human insulin was cloned and expressed in B. subtilis by joining the first third of the B. licheniformis penicillinase gene with the preproinsulin gene. This resulted in the secretion of a fusion protein into the media that was detected by antibodies to insulin. The B. licheniformis penicillinase promoter was also used to express .beta.-interferon (IF) in B. subtilis. In this case, the B. licheniformis penicillinase gene including the signal peptide was removed, and the gene coding for the mature .beta.-IF (interferon) was fused at the 3' end of the penicillinase promoter. The resulting plasmid was used to transform B. subtilis cells. .beta.-interferon was detected within the B. subtilis cells that were transformed with this plasmid containing the .beta.-IF sequence.
It was also shown that a Bacillus signal peptide could direct the secretion of foreign protein (.beta.-lactamase) outside the cell in B. subtilis. Palva, I. et al., (1982) Proc. Nat. Acad. Sci USA 79, 5582-5586 used the .alpha.-amylase promoter and signal sequence from Bacillus amyloliquefaciens (herein "B. amyloliquefaciens") to successfully express and secrete E. coli .beta.-lactamase in B. subtilis. The amylase promoter and signal was joined to the start of the mature E. coli gene coded for .beta.-lactamase from which the E. coli .beta.-lactamase signal sequence was removed. Proper fusions to the mature gene resulted in the secretion and correct processing by B. subtilis of the .beta.-lactamase protein into the outside media.
The prior art has shown the use of B. licheniformis and B. amyloliquefaciens signals and promoters in B. subtilis cloning vectors to express and secrete foreign protein by B. subtilis.